Cytosolic GTP-bound Ras has been shown to act as a dominant negative (DN) inhibitor of Ras by sequestering Raf in non-productive cytosolic complexes. Nevertheless, this distinct class of DN mutants has been neither well characterized nor extensively used to analyze Ras signaling. In contrast, DN Ras17N, which functions by blocking Ras guanine nucleotide exchange factors, has been well characterized and is widely used. Cytosolic GTP-bound Ras mutants could be used to inhibit particular Ras effectors by introducing additional mutations (T35S, E37G or Y40C) that permit them to associate selectively with and inhibit Raf, RalGDS, or phosphoinositide 3-kinase, respectively. When the wild-type Ras effector binding region is used, cytosolic Ras should associate with all Ras effectors, even those that are not yet identified, making these DN Ras mutants effective inhibitors of multiple Ras functions. We generated cytosolic GTP-bound H-, N-, and K-Ras, and we assessed their ability to inhibit Ras-induced phenotypes. In fibroblasts, cytosolic H-, N-, and K-Ras inhibited Ras-induced Elk-1 activation and focus formation, induced a flattened cell morphology, and increased adhesion to fibronectin through modulation of a  1 -subunit-containing integrin, thereby demonstrating that DN activity is not limited to a subset of Ras isoforms. We also generated cytosolic GTP-bound Ras effector domain mutants (EDMs), each of which reduced the ability of cytosolic GTP-bound Ras proteins to inhibit Elk-1 activation and to induce cell flattening, implicating multiple pathways in these phenotypes. In contrast, Ras-induced focus formation, platelet-derived growth factor (PDGF)-, or Rasinduced phospho-Akt levels and cell adhesion to fibronectin were affected by T35S and Y40C EDMs, whereas PDGF-or Ras-induced phospho-Erk levels were affected only by the T35S EDM, implying that a more limited set of Ras-mediated pathways participate in these phenotypes. These data constitute the first extensive characterization of this functionally distinct class of DN Ras inhibitor proteins.The small GTPase, Ras, is involved in numerous aspects of normal cellular metabolism including proliferation, survival/ apoptosis, differentiation, and adhesion/motility (1). Ras must be localized to the inner leaflet of the plasma membrane where, in its GTP-bound active form, it translocates cytosolic effector proteins such as Raf kinase, Ral guanine nucleotide dissociation stimulator (RalGDS), 1 or phosphoinositide 3-kinase (PI3-K) to the membrane for subsequent activation. Ras is anchored at the membrane as the result of a series of posttranslational modifications beginning with the attachment of a farnesyl isoprenoid to a conserved cysteine residue (cysteine 186 in H-and N-Ras; cysteine 185 in K-Ras) located within the "CAAX box," a motif consisting of the carboxyl-terminal four amino acids of Ras. Mutation of the CAAX cysteine therefore prevents both Ras farnesylation and membrane association, leaving the Ras protein unprocessed in the cytosol, where the mutationally ac...